T cell Dependent Bispecific (TDB) antibodies are designed to bind a target antigen expressed on a cell, and to bind to T cells, often by binding CD3e subunit of the T cell receptor. The binding of the bispecific antibody to the extracellular domains of both the target antigen and to the CD3 of T cell results in T cell recruitment to target cells resulting in T cell activation and target cell depletion. In the absence of the target cell, the single anti-CD3 arm is not able to cross-link TCRs to induce T cell activation and target cell killing. Anti-CD3 homodimer is a product related impurity that is formed during the manufacturing process of TDB antibodies and is capable of cross-linking TCR and inducing a low level of T cell activation in the presence or absence of target cells. Anti-CD3 homodimer may also impact therapeutic efficacy if present at high levels which can result in a decrease in TDB biological potency in vitro. Anti-CD3 homodimer can have off-target effects by inducing a low level of T cell activation and inflammatory cytokines by T cells in the absence of target cells. It is therefore desirable to control the levels of T cell activating product related variants present in the manufacturing process of TDB and a sensitive, reproducible and quantitative impurity assay method is needed to detect anti-CD3 homodimers that may be present in the purified product in order to support the development of a safe and efficacious clinical drug candidate.
Impurity assays need to be able to distinguish between the product/process related impurity and the desired product. Many traditional approaches for Chinese Hamster Ovary Cell protein (CHOP) impurity detection use a binding assay format approach, where the presence of process related CHO proteins can be sensitively detected in the product to evaluate product purity and safety. These CHOP antibodies are specific for the CHOP proteins, but do not recognize the product, so there is in general no impact to sensitively detecting impurities in the presence of the final product. Similar approaches could be used for bi-specific antibodies, provided antibodies can be identified that would distinguish between the final product and the impurity. The implementation of a useful anti-CD3 homodimer binding assay format would require the development of highly specialized antibodies, which may not be possible for this antigen, or other bi-specifics in general. Alternative physiochemical based methods (RP-HPLC, Mass Spec) can also be used to detect product related impurities and rely on the ability to adequately separate product related impurities from the product, and thereby detect the amount of impurity present. The amount of the impurity is detected relative to the other species present in the material, or by spiking in a variant standard and comparing the percent of material present to the spiked standard. However, many of these methods may involve additional sample handling and processing steps in order to separate the variant from the desired product material and these steps may alter the material or limit the sensitivity and accuracy of the method. Moreover, it is also desirable to know that the structural isoforms of any anti-CD3 homodimer product related impurity, which may be present in the bi-specific Test Article (purified product, DS, DP, stability sample, stress sample), or other potential T cell activating impurities, are biologically active in order to assign appropriate risk to the impurity. The novel anti-CD3 homodimer assay approach described herein uses a cell-based approach to detect biologically active anti-CD3 homodimer impurities and thereby avoids the challenges and limitations for homodimer impurity detection in bi-specific preparations using binding assay or physiochemical based formats.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.